Botulinum poisoning is a serious and well-recognized biothreat. Botulinum neurotoxins (BoNT) are extremely potent, relatively simple to prepare/disburse, and have already been used as a bioterror agent. There is a vital need for sensitive and specific methods for early detection of BoNT in the blood of exposed individuals before clinical symptoms have progressed to a critical state. The current "gold standard" for laboratory diagnosis of botulism is the mouse bioassay. While sensitive, it is also slow (2-4 days) and requires a dedicated animal laboratory. This underscores the unmet need for a rapid, sensitive and easy to use BoNT detection technology. The overall goal of the project is to build a sensor for rapid (30~60 minutes) in-vitro detection of BoNT/A and BoNT/B in serum. The sensor will be aimed at use in diagnostic laboratories and public health facilities. The sensor will be built based on a proven waveguide implementation of the surface plasmon resonance technology. It will use enzymatic cleavage assays for BoNT/A and BoNT/B. A peptide substrate will be synthesized, including the cleavage sequences for BoNT/A and BoNT/B. Gold nanoparticles will be attached to the peptide to increase the SPR detection sensitivity. An immuno-affinity based sample protocol will be developed to extract and concentrate the target BoNTs from serum.